ES2939643T3 - Rep protein as a protein antigen for use in diagnostic assays - Google Patents

Abstract

Disclosed is a DNA replication associated protein (Rep) for use in the diagnosis of multiple sclerosis (MS), wherein (a) an increased amount of Rep protein or fragments thereof in a sample from a subject compared to with an amount in a control sample; or a greater amount of anti-Rep protein antibodies to antigen in a sample from a subject compared to an amount in a control sample correlates with a diagnosis of MS, where the Rep protein is an MSBI1 or MSBI2 Rep. (Automatic translation with Google Translate, without legal value)

Description

DESCRIPTION

Rep protein as a protein antigen for use in diagnostic assays

The invention relates to methods for diagnosing multiple sclerosis (MS) in which at least 2-fold greater amount of a ^{D n} replication-associated protein (Rep) or at least 2-fold greater amount of anti- Rep in a sample from a subject compared to an amount in a control sample correlates with a diagnosis of multiple sclerosis (MS). In particular, the invention relates to a Rep protein encoded in the MSBI1 genome. The invention is defined by the appended claims. Embodiments and descriptions that no longer fall within the scope of the appended claims are merely considered examples suitable for understanding the invention.

The etiology of multiple sclerosis (MS) has not been resolved. Therefore, there is a demand for a biomarker for MS that can be used for the diagnosis of MS and/or the monitoring of MS or a treatment of MS and/or assessing a predisposition to MS.

Multiple sclerosis (MS) is characterized by demyelination of lesions that damage nerve cells in the brain and spinal cord. MS symptoms present as sudden worsening episodes (relapses, exacerbations, flare-ups, attacks) or as gradual worsening over time (progressive forms). Demyelination initiates inflammatory processes that trigger T cells and the release of cytokines and antibodies. Neuroimaging, cerebrospinal fluid analysis, and evoked potentials are used to diagnose MS.

A spectrum of 17 different, though partially related, DNA molecules was isolated from different test materials (multiple sclerosis (MS) brain tissue, bovine serum, milk) (Funk, Gunst et al. 2014, Gunst, Zur Hausen et al. 2014, Lamberto, Gunst et al. 2014, Whitley, Gunst et al. 2014). Among these isolates, two DNA molecules closely related to the isolate Sphinx 1.76 (1,758 bp; access no. HQ444404, (Manuelidis L. 2011)), associated with transmissible spongiform encephalopathy (TSE), were isolated from brain tissue of MS patients. These isolates were MSBI1,176 (MSBI, Multiple Sclerosis Brain Isolate) (1,766 bp) and MSBI2,176 (1,766 bp) which are designated "MSBI1 genome" and "MSBI2 genome", respectively. MSBI1,176 shares 98% nucleotide similarity with the Sphinx 1.76 sequence. The large open reading frames (ORFs) of the isolates encode a putative DNA replication protein that shares great similarity between them. Another common feature is the presence of iteron-like tandem repeats. Alignment of this region of repeats indicates individual nucleotide core variation. These iteron-like repeats may constitute the binding sites of Rep proteins. The site of the isolates has been deposited in the EMBL data bank under accession numbers LK931491 (MSBI1.176) and LK931492 (MSBI2.176) (Whitley C et al., 2014) and have been aligned and described in WO 2016/005064.

Other isolates were obtained from cow's milk. These cow's milk isolates (CMI) were CMI1.252, CMI2.214 and CMI3.168, which are designated as "CMI1 genome", "CMI2 genome" and "CMI3 genome", respectively. The sequences of the isolates have been deposited in the EMBL data bank under the accession numbers LK931487 (CMI1.252), LK931488 (CMI2.214) and LK931489 (CMI3.168) and have been aligned and described in the document WO 2016 /005064.

EP 2868751 describes a method for detecting a polypeptide encoded by an HCBI polynucleic acid.

EP 2966176 searches for nucleic acids in human and animal samples that resemble viral or bacterial pathogens and reveals the isolate MSBI1.176. MSBI1.

The present inventors have discovered that the MSBI1 genome shows significant production of RNA transcripts and that the Rep protein encoded in the MSBI1 genome is expressed in human cells. The present inventors have discovered that the Rep protein encoded in the MSBI1 and MSBI2 genome (MSBI1 Rep and MSBI2 Rep) represents a biomarker for pathogenicity screening assays. As a DNA replication associated protein (RepB), the Rep protein has DNA binding activity and may be essential for the initiation of replication of episomal or viral DNA molecules. The Rep proteins, which are structurally similar to the Rep encoded in the MSBI1 genome according to the present invention, show a marked potential for autooligomerization and aggregation, which was described in prokaryotic systems in vivo and in vitro (Giraldo, Moreno-Díaz de la Espina et al. 2011, Torreira, Moreno-Del Álamo et al. 2015).

The present invention provides a platform for pathogen-specific diagnostic screening assays based on the use of a Rep protein as antigen.

In certain embodiments, anti-Rep antibodies are used as pathogenic markers due to the linkage of the pathogenic activity of the DNA isolate (MSBI1) to expression of the Rep protein. Patient sera containing elevated amounts of anti-Rep antibodies indicate that the patient The corresponding counterpart was definitely exposed to Rep-related or Rep-expressed proteins for a period of time long enough to initiate a Rep-specific immune response. The Rep protein is used as the antigen as the target for human antibodies. Based on the quantification of the amount of anti-Rep antibodies, acute MS can be diagnosed or monitored, as well as the predisposition to MS. Since an increase in the amount of induced anti-Rep antibodies or expressed Rep protein in a sample has been recognized as indicating the onset and/or stage of MS, an increase in the amount of anti-Rep antibodies and protein Rep, respectively, can be used as a pathogenic biomarker for the diagnosis of MS.

Advantageously, the pathogenic biomarker of MS can be detected in blood samples, such as serum or plasma, and it is not necessary to obtain samples of the cerebrospinal fluid.

Therefore, the invention provides a DNA replication associated protein (Rep) for use in the diagnosis of multiple sclerosis (MS). The Rep protein is encoded by the MSBI1 genome and comprises (i) an amino acid sequence as represented in SEQ ID NO:1;

(ii) a fragment of SEQ ID NO:1 that is capable of binding to an anti-Rep antibody specific for a protein comprising an amino acid sequence as described in s Eq iD NO:1; or (iii) an amino acid sequence that is 90% or more homologous to the amino acid sequence of (i) or (ii) and is capable of binding an anti-Rep antibody specific for a protein comprising an amino acid sequence as depicted in SEQ ID NO:1.

Another Rep protein that is not part of the claimed invention is encoded by the MSBI2 genome and comprises (i) an amino acid sequence as represented in SEQ ID NO:8;

(ii) a fragment of SEQ ID NO:8 that is capable of binding to an anti-Rep antibody specific for a protein comprising an amino acid sequence as described in ^s E ^q iD NO:8; or (iii) an amino acid sequence that is 90% or more homologous to the amino acid sequence of (i) or (ii) and is capable of binding an anti-Rep antibody specific for a protein comprising an amino acid sequence as depicted in SEQ ID NO:8.

Another Rep protein that is not part of the claimed invention is encoded by the CMI1 genome and comprises (i) an amino acid sequence as represented in SEQ ID NO:10;

(ii) a fragment of SEQ ID NO:10 that is capable of binding to an anti-Rep antibody specific for a protein comprising an amino acid sequence as depicted in ^s E ^q ID ⁿ O:10; either

(iii) an amino acid sequence having 90% or more homology to the amino acid sequence of (i) or (ii) and capable of binding an anti-Rep antibody specific for a protein comprising an amino acid sequence such as that represented in SEQ ID NO:10.

Another Rep protein that is not part of the claimed invention is encoded by the CMI2 genome and comprises (i) an amino acid sequence as represented in SEQ ID NO:11;

(ii) a fragment of SEQ ID NO:11 that is capable of binding to an anti-Rep antibody specific for a protein comprising an amino acid sequence as described in SEQ ID NO:11; or (iii) an amino acid sequence that has 90% or more homology to the amino acid sequence of (i) or (ii) and is capable of binding an anti-Rep antibody specific for a protein comprising a sequence of amino acids as depicted in SEQ ID NO:11.

Another Rep protein that is not part of the claimed invention is encoded by the CMI3 genome and comprises (i) an amino acid sequence as represented in SEQ ID NO:12;

(ii) a fragment of SEQ ID NO:12 that is capable of binding an anti-Rep antibody specific for a protein comprising an amino acid sequence as represented in SEQ ID NO:12; or (iii) an amino acid sequence having 90% or more homology to the amino acid sequence of (i) or (ii) and capable of binding to an anti-Rep antibody specific for a protein comprising a sequence of amino acids as depicted in SEQ ID NO:12.

A greater amount of Rep protein in a sample from a subject compared to an amount of Rep protein in a control sample correlates with a diagnosis of MS, ie, indicates MS. In accordance with the present invention, the diagnosis of MS or a predisposition to MS is indicated by an increase in the amount of Rep protein of at least 2-fold compared to a control sample.

In other embodiments, an increase in the amount of anti-Rep antibodies in a sample from a subject compared to the amount of anti-Rep antibodies in a control sample correlates with a diagnostic of MS, that is, it is indicative of MS. According to the present invention, the diagnosis of MS or predisposition to MS is indicated by an increase in the amount of anti-Rep antibodies of at least 2-fold compared to a control sample.

The Rep protein of the present invention can be used in virtually any assay format that employs a known antigen to detect antibody or cell-mediated immune responses. Thus, the present invention also encompasses the detection of cell-mediated immune responses, eg, T cells, against the Rep protein.

The invention provides a method of diagnosing MS in a subject comprising the steps of

(a) incubating a sample obtained from a subject with a Rep protein;

(b) detecting the amount of antibodies in the subject's sample that form an immune complex with the Rep protein; and

(c) correlating at least a 2-fold higher amount of antibodies bound to the Rep protein, compared to an amount in a control sample, with a diagnosis of MS.

In particular embodiments, the Rep protein is immobilized, eg, attached to a scaffold or carrier, followed by incubation of the immobilized Rep protein with the subject's sample.

In other embodiments, the Rep protein is expressed in cells and the cells are then incubated with the subject's sample.

In certain embodiments, the amount of antibodies forming an immune complex with the Rep protein is quantitated by an additional binding agent coupled to a signal-generating compound capable of binding to the anti-Rep antibodies in the immune complex, e.g., an antibody detectably labeled secondary, preferably anti-human antibody.

In other embodiments, antibodies from the subject sample are immobilized and then incubated with a defined amount of Rep protein.

The sample from the subject and the control sample is a blood sample, such as serum or plasma.

The invention provides a method of diagnosing MS in a patient comprising the steps of

(a) detecting the amount of Rep protein in a sample from a subject by anti-Rep antibodies that bind to an epitope comprised by SEQ ID NO:2 or SEQ ID NO:3; and

(b) correlating at least a 2-fold greater amount of Rep protein detected in the sample from a subject in step (a) compared to an amount in a control sample diagnosed with MS.

In such embodiments, a subject's sample and a control sample are selected from the group consisting of a serum sample, a plasma sample, or a tissue sample.

In particular embodiments, the anti-Rep antibody binds to an epitope that is within an amino acid sequence selected from the group consisting of amino acids 1-136, 137-229, and 230-324 of SEQ ID NO:1. .

In other embodiments, the invention provides a kit for use in the diagnosis of MS comprising (a) an MSBI1 Rep protein, (b) an anti-human antibody according to Table 1 coupled to a detectable tag and capable of binding to the anti-human antibody. -Rep according to the invention, and (c) a solid matrix suitable for immobilizing a Rep protein according to (a) or anti-Rep antibodies, where the helper antibodies are suspected in a sample, in particular a serum or plasma sample.

In certain embodiments, the kit is prepared for use in an immunoassay, eg, selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), an enzyme immunoassay (EIA), a fluorescence immunoassay (FIA). ), a luminescence immunoassay (LIA) and a strip assay.

Brief description of the drawings:

Figure 1 shows an example of screening 2*8 sample serum by incubating serum from 1D size resolved Rep protein membrane strips. Nitrocellulose strips containing Rep protein were incubated with two different serum/plasma dilutions (1:500 and 1:2000) from either the MS serum sample pool or the healthy donor plasma pool. Rep-bound human IgG was detected with HRP-coupled anti-human IgG secondary antibodies. The signs of the antibodies to the full-length Rep protein target size (see Coomassie protein staining and anti-Rep WB controls at right) were quantified by densitometry.

Figure 2 shows a quantification of Rep-specific sero-responses based on ELISA screening. 50, 100, 200, 400 or 800 ng of Rep protein (target antigen) were placed per 96 wells. After blocking and washing, the Rep protein antigen was incubated with an MS serum sample or a control plasma sample at a dilution of 1:500 for 6 hours at room temperature (RT). After washing and incubation with a HRP-coupled anti-human IgG secondary antibody and a final washing step, the presence of Rep-bound human IgG was quantified by reaction with TMB substrate and measurement of absorbance at 450 nm. Quantification reveals a good correlation between signal intensity and the amount of Rep protein (antigen). On average, the EM pool signal intensity levels exceed the control pool intensities by at least a factor of 1.9. In general, ELISA-based serum screening revealed detectable antibodies in serum to at least a 1:1000 dilution of serum (data not shown), so it is likely that the titer for this experiment is greater than 1:1000 in the region of 1:2000-1:4000 or even higher after platform optimization.

Figure 3 shows a quantification of Rep-specific sero-responses based on ELISA screening. 200 ng of Rep protein (target antigen) was placed per 96 wells. After blocking and washing, the Rep protein antigen was incubated with 7 individual MS sera or 7 individual control sera in duplicate at a dilution of 1:500 for 6 h at room temperature (RT). After washing and incubation with a HRP-coupled anti-human IgG secondary antibody and a final washing step, the presence of Rep-bound human IgG was quantified by reaction with TMB substrate and measurement of absorbance at 450 nm. The mean intensities of the EM samples exceed the mean intensity of the control sera by at least a factor of 8, with some EM samples revealing a factor of 10-16 over the mean control intensities.

Figures 4 to 6 show immunofluorescence imaging data obtained using the anti-Rep A, B, C, D1 and D2 antibodies which are designated at the "y" coordinates.

Figure 7 shows a Western Blot analysis, in which A, B, C and D1 designate the groups of anti-Rep antibodies used.

Figure 8 shows a sequence alignment of the "optimized" (=SEQ ID NO:13) vs. "original" (=wild-type; SEQ ID NO:12) MSBI1 sequence. Optimized codons are marked.

The invention provides diagnostic screening assays for the presence of anti-Rep antibodies as pathogenic markers. Samples containing elevated amounts of anti-Rep antibodies indicate that the corresponding subject was either definitely exposed to the Rep-related protein or expressed the Rep protein for a period of time long enough to induce a Rep-specific immune response. Rep protein. With such screening assays, diagnosis, prognosis and monitoring of MS based on the quantification of anti-Rep antibodies can be performed. In alternative embodiments, the Rep protein can be directly detected and quantified in samples using anti-Rep antibodies.

"Rep Protein" as used herein refers to a DNA replication associated protein (RepB). The Rep protein comprises DNA binding activity and might be essential for the initiation of replication of episomal/viral DNA molecules. In general, Rep protein refers to a Rep protein from the Small Sphinx genome group (Whitley et al., 2014). The Rep protein according to the invention is a Rep protein encoded in the MSBI1 genome (MSBI1 Rep). A Rep protein encoded in the MSBI2 genome (MSBI2 Rep), a Rep protein encoded in the CMI1 genome (CMI1 Rep), a Rep protein encoded in the CMI2 genome (CMI2 Rep) or a Rep protein encoded in the CMI1 genome (CMI2 Rep) does not form part of the claimed invention. in the CMI3 genome (CMI3 Rep). Preferably, the MSBI1 Rep protein is encoded by MSBI1.176 deposited in the EMBL data bank under acc. no. LK931491 and has the amino acid sequence represented in NO. ID. SEQ. :1 (Whitley, Gunst et al. 2014). The CMI1 Rep protein encoded by CMI1.252, deposited in the EMBL data bank under acc. no. LK931487 and having the amino acid sequence represented in NO. ID. SEQ. :10; the CMI2 Rep protein encoded by CMI2.214, deposited in the EMBL data bank under acc. no. LK931488 and having the amino acid sequence described in NO. ID. SEQ. :eleven; the CMI3 Rep protein encoded by CMI3.168, deposited in the EMBL data bank under acc. LK931489 and having the amino acid sequence represented in NO. ID. SEQ. :12. In a particular preferred embodiment, the Rep protein comprises an N-terminal region conserved among small Sphinx genomes consisting essentially of amino acids 1 to 229 of NO. ID. SEQ. :1 and a C-terminal variable region specific for MSBI1.176 consisting essentially of amino acids 230 to 324 of NO. ID. SEQ. :1. The N-terminal conserved region comprises a first putative DNA-binding domain consisting essentially of amino acids 1 to 136 of NO. ID. SEQ. :1 and a second putative domain binding to DNA consisting essentially of amino acids 137 to 229 of NO. ID. SEQ. :1.

The "Rep protein" also encompasses fragments and variants of the NO protein. ID. SEQ. :1 that are capable of binding an anti-Rep antibody specific for the Rep protein having the amino acid sequence of NO. ID. SEQ. :1. Preferably, said fragment is an immunogenic fragment of the protein having the amino acid sequence of NO. ID. SEQ. :1 encompassing at least one epitope for an anti-Rep protein antibody against the Rep protein of NO. ID. SEQ. :1 and preferably comprises at least 7, 8, 9, 10, 15, 20, 25 or 50 contiguous amino acids. In particular embodiments, the fragment comprises or consists essentially of a domain of the Rep protein, eg, the N-terminal conserved region, the C-terminal variable region, the first or second DNA-binding domain. A variant of the protein with NO. ID. SEQ. :1 comprises one or more amino acid deletions, substitutions or additions compared to NO. ID. SEQ. :1 and has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequence of NO. ID. SEQ. :1, wherein the variant is capable of binding to an anti-Rep antibody specific for a Rep protein having the amino acid sequence of NO. ID. SEQ. :1. Included within the definition of variant are, for example, polypeptides containing one or more analogues of an amino acid (including, for example, non-natural amino acids, peptide nucleic acid (PNA), etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both of natural and non-natural origin. The term Rep protein includes fusion proteins with a heterologous amino acid sequence, with a leader sequence or with a Tag sequence, and the like. In certain embodiments of the invention, the protein tags are genetically grafted onto the Rep protein described above. In particular, at least one protein tag binds to a polypeptide consisting of an amino acid sequence as described in NO. ID. SEQ. s:1.14. Said protein tags can be removed by chemical agents or by enzymatic means. Examples of protein tags are affinity tags or chromatography for purification. For example, the Rep protein can be fused to a Tag sequence, eg, selected from the group consisting of His6-Tag (Seq Id No:4), -T7-Tag (S Seq Id No:5), Label-Flag (No. Id. Sec.:6) and Label-Strep-II (No. Id. Sec.:7). to His-Tag (Sec. Id. No.: 4), to T7-Tag (Sec. Id. No.: 5), Flag-Tag (Sec. Id. No.: 6) or Strep-II-Tag ( No. Id. Sec.:7). Furthermore, fluorescence tags such as green fluorescence protein (GFP) or its variants can be attached to a Rep-protein according to the invention.

In a particular preferred embodiment, the Rep protein encoded in the MSBI1 genome (MSBI1 Rep) is codon-optimized for production in human cell lines (eg HEK-293, HEK293TT, HEK293T, HEK293FT, HaCaT, HeLa, SiHa, CaSki, HDMEC, L1236, L428, BJAB, MCF7, Colo678, any primary cell line) as well as bovine (eg MAC-T) or murine (eg GT1-7) cell lines. In terms of codon optimization, the nucleotide sequences encoding the antigens can be designed to employ codons that are used in the genes of the subject in which the antigen is to be produced. In a preferred embodiment, the codons used are "humanized" codons. This codon usage provides for efficient expression of antigens in human cells. Any suitable method of codon optimization may be used. Such methods and the selection of such methods are well known to those skilled in the art. According to the present invention, said codon-optimized variant of the Rep protein encoded in the MSBI1 genome (MSBI1 Rep) comprises or has the sequence described in SEQ ID. No.: 13 and comprises or has the amino acid sequence depicted in SEQ ID. In total, 686 nucleotides of the 927 nucleotide MSBI1 Rep gene encoding the Rep protein (from start codon to stop codon) were replaced to improve codon usage. A perfect codon usage with optimal adaptation towards, for example, human codon usage is described by a Codon Adaptation Index (CAI) of 1 indicating that all codons are optimized for human expression. For the original MSBI1-encoded Rep gene, a CAI of 0.67 was calculated for the human system, which is well below the CAI of 0.8, which is considered the lower threshold for good codon matching. Following codon optimization of the MSBI1 rep gene, a CAI of 0.94 was calculated for the optimized DNA, indicating a near-optimal codon match for the human system. In particular, 18 very infrequently used codons were modified, most of which represented leucine-encoding codons with a very low frequency of use (<10%) in the human system. A sequence alignment of the NO codon-optimized sequence MSBI1 is shown in Figure 8. ID. SEQ. :13 vs. the wild-type MSBI1 sequence from NO. ID. SEQ. :fifteen.

The Rep protein of the invention, including Rep fragments and Rep variants defined above, can be prepared by classical chemical synthesis. The synthesis can be carried out in a homogeneous solution or in a solid phase. Polypeptides according to this invention can also be prepared by recombinant DNA techniques. An example for the production and purification of a Rep protein according to the invention is shown in Example 1.

"Subject" as used herein refers to a mammalian individual or patient, including murine, cattle, eg, bovine, ape, and human. Preferably, the subject is a human patient.

"Sample" as used herein refers to a biological sample encompassing liquid and solid samples. Liquid samples include blood fluids such as serum, plasma, and cerebrospinal fluid (CSF). Solid samples encompass tissue samples such as tissue cultures or biopsy samples.

The term "Correlates with", as used herein, refers to an amount, ie, level or titer, of anti-Rep antibodies and Rep protein, respectively, significantly correlated with a disease state of, MS. Correlation is determined by detecting the degree of difference in the amount present in a sample from a test subject and a control sample. By "control sample" is meant a single sample or an average of several, ie more than two, control samples. The control is taken from a healthy individual who has not been diagnosed with MS. Alternatively, correlation can be determined theoretically by detecting the degree of difference in the amount present in a sample for a test subject with a predetermined cut-off value. A cut-off value is a reference value with a statistically significant separation between different disease states, for example, between the healthy state and the diseased state. The cut-off value can be determined by statistical analysis of a sufficiently large panel of test samples from patients with a history of disease and samples from a healthy test group by statistical tests known in the art.

In certain embodiments, a diagnosis, for example, of MS, is indicated by at least a 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold amount of protein, ie, Rep protein and anti-Rep antibodies, respectively, in the subject sample compared to a control sample.

"Anti-Rep antibody" as used herein refers to an antibody that binds at a detectable level to the Rep protein in the methods of the invention whose affinity is stronger to the Rep protein of the invention than to a non-Rep protein. -Rep. Preferably, the affinity of the antigen for the Rep protein is at least 2-fold greater than background binding. In particular, the anti-Rep antibody is specific for the MSBI1 Rep having the amino acid sequence of NO. ID. SEQ. :1. In certain embodiments, the antibody is specific for MSBI1 Rep, MSBI2 Rep, CMI1 Rep, CMI2 Rep, and/or CMI3 Rep. In certain embodiments, the anti-Rep antibody binds to an epitope within amino acids 1 to 136 of the NO. ID. SEC.: 1.

A common feature of all assays is that the Rep protein is contacted with a sample suspected of containing antibodies against the Rep protein under conditions that allow the Rep protein to bind to any such antibody present in the sample. Such conditions will typically be physiological temperature, pH, and ionic strength using an excess of Rep protein. Incubation of Rep protein with the sample is followed by detection of immune complexes formed by the antigen. In certain embodiments, the Rep protein is coupled to a signal-generating compound, eg, a detectable label, or an additional binding agent, eg, an anti-human secondary antibody, coupled to a signal-generating compound is used to detect the immune complex.

Anti-Rep antibodies can be detected and quantified in assays based on the Rep protein as protein antigen, which serves as a target for mammalian, eg, human, antibodies suspected in the sample. Preferably, the Rep protein is purified (eg see Example 1) and the samples may be, for example, serum or plasma samples. The methods include immobilization of the Rep protein on a matrix followed by incubation of the immobilized Rep protein with the samples. Finally, Rep-bound antibodies to the immune complex formed between the Rep protein and the antibodies in the samples are quantitated using a detection binding agent coupled to a signal-generating compound, for example, an HRP-coupled secondary detection antibody. (radish-peroxidase) which allows a HRP substrate based quantification. This signal or label generating compound is detectable by itself or can be reacted with an additional compound to generate a detectable product.

In other embodiments, anti-Rep antibodies are quantitated indirectly in that the sample's antibodies are first immobilized on a matrix, followed by incubation with a defined amount of Rep protein that may be labeled or comprise a Tag protein, in which the antibodies immobilized anti-Rep present on the matrix capture Rep protein from the liquid protein-sample mixture, followed by quantification of bound Rep protein.

In other embodiments, the Rep protein can be expressed in cells and these cells are incubated with the sample. Anti-Rep antibodies in the sample bound to the Rep protein expressed by the cells are then detected and quantified.

Immunoassay design is subject to great variation, and many formats are known in the art. Protocols can, for example, use solid supports or immunoprecipitation. Most assays involve the use of binding agents coupled with signal-generating compounds, eg, labeled antibodies or labeled Rep proteins; the labels can be, for example, enzymatic, fluorescent, chemiluminescent, radioactive or dye molecules. Assays that amplify immune complex signals are also known; examples of which are assays using biotin and avidin or streptavidin, and enzyme-mediated and labeled immunoassays, such as ELISA assays.

The immunoassay can be of a heterogeneous or homogeneous format, and of a standard or competitive type. In a In heterogeneous format, the polypeptide (Rep protein or anti-Rep antibody) is typically attached to a solid matrix or support or carrier to facilitate sample separation from the polypeptide after incubation. Some examples of solid supports that can be used are nitrocellulose (for example, in the form of membrane or microtiter wells), polyvinyl chloride (for example, in sheets or microtiter wells), polystyrene latex (for example, in microtiter beads or plates, polyvinylidine fluoride (known as Immunolon), diazotized paper, nylon membranes, activated beads, and protein A beads. The solid support containing the antigenic polypeptides is usually washed after separation from the test sample and prior to detection of bound anti-Rep antibodies Standard and competitive formats are known in the art.

In a homogeneous format, the test sample is incubated with the Rep protein in solution. For example, it can be done under conditions that precipitate any Rep protein-antibody complexes that form. Both standard and competitive formats for these assays are known in the art.

In a standard format, the amount of anti-Rep antibodies in the antibody-Rep protein complexes is directly monitored. This can be accomplished by determining whether (labelled) antixenogeneic (eg, anti-human) antibodies that recognize an epitope on the anti-Rep antibodies will bind due to complex formation. In a competitive format, the amount of anti-Rep antibodies in the sample is inferred by monitoring the competitive effect on the binding of a known amount of labeled antibody (or other competing ligand) to the complex.

Complexes formed comprising anti-Rep antibody (or in the case of competitive assays, the amount of competing antibody) are detected by any of a number of known techniques, depending on the format. For example, unlabeled anti-Rep antibodies in the complex can be detected using an anti-xenogeneic Ig conjugate complexed with a tag (eg, an enzyme tag, such as HRP).

In an immunoprecipitation or agglutination assay format, the reaction between the Rep protein and the anti-Rep antibody forms a network that precipitates from solution or suspension and forms a visible layer or film of precipitate. If no anti-Rep antibody is present in the sample, no visible precipitate forms.

The selected solid phase can include polymeric or glass beads, nitrocellulose, microparticles, microwells of a reaction tray, test tubes, and magnetic beads. The signal-generating compound may include an enzyme, a luminescent compound, a chromogen, a radioactive element, and a chemiluminescent compound. Some examples of enzymes are alkaline phosphatase, horseradish peroxidase (HRP), and beta-galactosidase. Some examples of enhancer compounds are biotin, antibiotin, and avidin. Examples of binding members of enhancer compounds include biotin, antibiotin, and avidin.

In other embodiments, the invention provides methods in which an increased amount of Rep protein in a sample is correlated with a diagnosis or predisposition for MS.

In such embodiments the Rep protein in the sample is detected by anti-Rep antibodies.

Such methods comprise the steps of

(a) detecting the amount of Rep protein in a sample from a subject by anti-Rep antibodies; and

(b) correlating the amount of Rep protein detected in the sample from a subject in step (a) compared to an amount in a control sample with a diagnosis of MS.

Examples of assays that can be used in such methods for the detection of Rep protein in serum or plasma samples include, but are not limited to, immunoprecipitation, immunofluorescence, dot blotting, and Western blotting.

For example, a serum sample can be incubated with antibodies to the Rep protein to capture the Rep protein in the sample, followed by a Rep protein immunoprecipitation step, and then a detection step by SDS-PAGE and Western Blotting. .

In another example, a dot blot membrane can be incubated with serum, followed by the SDS-PAGE and Western Blot step.

In another example, serum dilutions of the sample are loaded onto SDS-Page followed by Western blotting.

In other embodiments, Rep protein is detected in tissue samples by immunohistochemical methods or immunofluorescence microscopy.

In certain embodiments, anti-Rep antibodies are used for the detection or capture of the Rep protein in the sample.

The term "antibody" preferably refers to antibodies that consist essentially of pooled polyclonal antibodies with different epitopic specificities, as well as different preparations of monoclonal antibodies. As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) includes intact immunoglobulin molecules as well as antibody fragments (such as Fab and F(ab') fragments). 2) capable of specifically binding to the Rep protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, are cleared more rapidly from the circulation, and may have less non-specific tissue binding than intact antibody. Therefore, these fragments are preferred, as well as the products of an expression library of FAB or other immunoglobulins. In addition, antibodies useful for the purposes of the present invention include chimeric, single chain, multifunctional (eg, bispecific), and humanized antibodies or human antibodies.

In certain embodiments, the antibody or antigen-binding fragment thereof is coupled to a signal-generating compound, eg, carries a detectable label. The antibody or antigen-binding fragment thereof can be directly or indirectly detectably labeled, for example, with a radioisotope, fluorescent compound, bioluminescent compound, chemiluminescent compound, metal chelator, or enzyme. Other suitable tags for binding to the antibody will be known to those skilled in the art or will be able to determine them through routine experimentation.

Anti-Rep antibodies are preferably raised (generated) against a Rep protein having the amino acid sequence of NO. ID. SEQ. :1 or a fragment thereof by methods well known to those skilled in the art.

In certain embodiments, anti-Rep antibodies are used in the methods of the invention that are capable of binding to several or all types of Rep proteins from the Little Sphinx Genome cluster (anti-Little Sphinx Rep antibody or anti-SSLRep antibody). ). Said anti-SSLRep antibody binds to an epitope ^{within the conserved N-terminal region of the Rep protein from amino acids 1 to 229 of NO. ID. s} E ^c . ^:1. In particular embodiments, anti-Rep antibodies of the anti-SSLRep type that bind to an epitope within NO are used. ID. SEQ. :2 (amino acids 32-49 of SEQ ID NO:1) or NO. ID. SEQ. :3 (amino acids 197-216 ^{of SEQ ID NO:1). The peptide fragments of NO. ID.} S ^{and C.:2 and NO. ID.} S ^and C. ^{:3 are highly} conserved among the Rep proteins of the Little Sphinx Genome group and appear to be exposed due to their hydrophilic character. Anti-Rep antibodies of the anti-SSLRep type can be produced by immunization, for example of mice or guinea pigs, with peptides consisting essentially of the amino acid sequences as represented by NO. ID. SEQ. s:2 or 3; or by other immunogenic fragments, preferably comprising at least 8-15 amino acids, derived from the conserved N-terminal region of the Rep protein from amino acids 1 to 229 of NO. ID. SEQ. :1.

Anti-Rep antibodies specific for the Rep protein MSBI1 are used. Such antibodies can be produced, for example, by immunizing a mammal, such as mice or guinea pigs, with a full-length Rep protein having the amino acid sequence of NO. ID. SEQ. :1.

The methods of the invention use anti-Rep antibodies that are capable of detecting Rep protein down to picogram to femtogram ranges in different types of body fluids such as blood, serum, cerebrospinal fluid, or brain fluid.

A specific type of anti-Rep antibody or a set of two or more different types of anti-Rep antibodies may be used in the methods of the invention. If a pool of different types of anti-Rep antibodies is used, the pool of anti-Rep antibodies may comprise different anti-Rep antibodies that bind to different epitopes within different domains of the Rep protein, eg, first binding domain. to DNA (eg, aa 1-136 of SEQ ID NO:1), second ^{DNA -binding domain (eg, aa 137-229 of} SEQ ^{ID NO:2), and/ or variable domain (eg, aa 230-324 of SEQ ID NO:1), in particular, from the} Rep protein MSBI1 (SEQ ID NO:1).

In another embodiment, the anti-Rep antibodies are used for the screening of probes from patients and/or healthy individuals for a Rep protein. Selective detection of a Rep protein in patient tissues, for example, of a neurodegenerative disease, is indicative of a causality between the DNA genome isolated from the Rep protein detected in the sample and the disease of the patient from whom the sample comes.

For the detection of a Rep protein by means of anti-Rep antibodies, methods such as Western blotting, immunofluorescence microscopy or immunohistochemical methods can be applied.

In certain embodiments, anti-Rep antibodies capable of detecting a Rep protein in certain cell locations. For example, the anti-Rep antibody can detect Rep protein in the cytoplasm, nuclear membrane, and nucleus or detect specks in the cytoplasm. Examples of such groups of anti-Rep antibodies are shown in Table 1:

Anti-Rep group A antibodies have an epitope within the amino acid sequence depicted ^{in NO. ID.} S ^{e C.:3 (aa 198-217 of SEQ ID NO:1) and are capable of detecting MSBI1 Rep and Rep proteins} comprising this conserved epitope from the Little Sphinx Genome cluster (eg, MSBI2, ^{CMI1, c} MI4). ^{In immunofluorescence assays such anti-Rep antibodies detect a specific pattern of} Rep localization, in which the major localization is homogeneously distributed over the cytoplasm and nuclear membrane; and an additional weak and homogeneously distributed localization is observed in the nucleus. An example of such a group A antibody is the antibody AB01 523-1-1 (DSM ACC3327) which was used in the examples as the group A antibody.

Anti-Rep group B antibodies have an epitope within the amino acid sequence depicted in NO. ID. SEQ. :2 (aa 33-50 of SEQ ID NO:1) and are capable of detecting MSBI1 Rep and Rep proteins comprising this conserved epitope from the Little Sphinx Genome cluster (eg, MSBI2, CMI1, CMI4) . In immunofluorescence assays such anti-Rep antibodies specifically detect specks (cytoplasmic aggregations) of the Rep protein (often at the periphery of the nuclear membrane). An example of such a group B antibody is the antibody designated AB02304-4-1 (DSM ACC3328) which was used in the examples as the group B antibody.

Anti-Rep group C antibodies specifically detect a structural epitope of MSBI1 (SEQ ID NO:1). In immunofluorescence assays, such anti-Rep antibodies detect a Rep-specific localization pattern, in which the major localization is distributed homogeneously throughout the cytoplasm and nuclear membrane; and an additional weak and homogeneously distributed localization is observed in the nucleus. An example of such a group C antibody is the antibody MSBI1 381-6-2 (DSM ACC3329) which was used in the examples as the group C antibody.

Anti-Rep group D antibodies specifically detect a structural epitope of MSBI1 (SEQ ID NO:1), where as the MSBI1 961-2-2 antibody designated "D1" detects an epitope represented in NO. ID. ^s E ^c . ^{:9 (aa 281-287) in the C-terminal domain of MSBI1. The MSBI1 761-5-1 antibody (DSM ACC3328)} designated "D2" detects a 3D structural epitope of MSBI1 that is exclusively accessible under in vivo conditions and is not accessible on Western Blots. In immunofluorescence assays, these anti-Rep antibodies specifically detect specks (cytoplasmic aggregations) of the Rep protein (often at the periphery of the nuclear membrane.

In certain embodiments, anti-Rep antibodies from groups A, B, C or D; or a combination of anti-Rep antibodies from at least two different groups A, B, C, or D are used to determine the type of Rep protein localization in a probe and whether such Rep protein localization correlates with a pathogenic function . For example, if there are specks present. In certain embodiments, ie, methods or kits of the invention, at least one anti-Rep antibody selected from groups A and B is combined with at least one anti-Rep antibody selected from groups C and D. In particular embodiments, i.e., methods or kits of the invention, a group A anti-Rep antibody is combined with at least one other anti-Rep antibody selected from groups B, C, and D. For example, a group A anti-Rep antibody may be combined with other anti-Rep antibodies from groups C and D. Such combinations of anti-Rep antibodies from different groups increase the distinctiveness of the diagnostic evaluation, particularly for the diagnosis of MS. These group A, B, C or D antibodies are preferred for use in the diagnosis of MS.

The following antibodies were deposited at the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ) [German Collection of Microorganisms and Cell Cultures] on September 28, 2017:

AB01 523-1-1 antibody under DSM ACC3327;

AB02304-4-1 antibody under DSM ACC3328;

MSBI1 381-6-2 antibody under DSM ACC3329; and

MSBI1 761-5-1 antibody under DSM ACC3330.

The MSBI1 961-2-2 antibody has been deposited under DSM ACC3331 at DSMZ on October 6, 2017.

In other embodiments, a kit for use in the diagnosis of MS is provided. The kit may comprise material for detecting anti-Rep antibodies and/or Rep protein together with instructions for use of the ^{materials in assays for the diagnosis of eM. The kit may comprise one or more of the following} components: a biomarker according to the invention, ie, Rep protein and anti-Rep antibodies, eg, antibodies in Table 1, respectively; a signal generating compound, a solid matrix for fixing a capture agent, a diluent for the samples, a wash buffer. Signal generating compound refers to a detectable label that is coupled to an additional binding agent capable of binding to the biomarker of the invention or directly coupled to the biomarker of the invention.

The invention is further illustrated by, but not limited to, the following examples:

Example 1:

Purification of the MSBI1 Rep protein

A nucleotide acid molecule encoding the full-length Rep open reading frame (ORF) identified within the MSBI1 genome is cloned into an expression plasmid (pEXP5-CT, Invitrogen) allowing protein expression based on a translational system. in vitro without high yield E. coli cells (ExpresswayTM Cell-Free E. coli Expression System, Invitrogen). The synthesized Rep protein having the amino acid sequence of NO. ID. SEQ. :1 within the in vitro translation reaction is denatured by adding 20 reaction volumes of 8 M urea sample buffer pH 8.0 containing 100 mM NaH2PO4, 10 mM Tris HCl, pH 8.0, 5 mM imidazole. Subsequently, the Rep protein is purified under denaturing conditions (20 mM imidazole for washing and 300 mM imidazole for protein elution) from a C-terminal His6 tag fused to the Rep protein. The quality of purification it is determined by Coomassie protein staining and Western blotting with anti-Rep protein antibodies. The purity of the Rep protein is calculated densitometrically and is greater than 95%. The purified protein is used directly for ELISA-based serum screening or is subjected to SDS-Page followed by transfer to nitrocellulose membranes for serum incubation of 1D size resolved Rep protein membrane strips.

Example 2:

Generation of serum master plates

Serum samples are first aliquoted to reduce sample freeze-thaw cycles. A master plate is then established by generating a 1:1 dilution of serum in PBS pH 7.2 stabilized with a final concentration of 0.02% (w/v) sodium azide in a U-shaped 96-well plate. This plate is stable for at least 2 months when stored at 4°C and handled under sterile conditions. This plate is stable for at least 2 months when stored at 4°C and handled sterile, and is used as a template for further dilution of serum (1:20 final dilution) prior to the individual screening experiment.

Example 3:

Screening by incubation with serum of 1D size resolved Rep protein membrane strips

For screening assays based on incubating 1D resolved Rep protein membrane strips with serum, 20 µg of purified Rep protein are loaded onto Sigma Tru-PAGE 4-20% precast gels. The walls separating the gel pockets are removed prior to loading the gel to produce one large pocket and a single pocket for a size marker. After 1D size resolved SDS-PAGE, the protein is transferred to nitrocellulose membranes using a standard tank/wet blot transfer protocol. The protein on the membrane blot (essentially a full length Rep protein band at a height of approximately 40 kDa) is then visualized with PonceauS to check the quality of the blot and to prepare for cutting 2 mm thick membrane strips. width and height of the complete membrane resolved in 1D size. The strips are then blocked for 1 h with each 500 µl of serum-free blocking reagent (Genetex Trident universal blocking reagent).

The serum is then characterized from two different serum dilutions. The serum is incubated for 14 h (overnight) at 4 °C in a linear shaker.

The membrane strips are then washed with PBS-T pH7.2 0.1% Tween-20 for 3x5 min at room temperature. Detection of human IgG specifically bound to Rep within sera is performed by incubation with a secondary antibody to goat anti-human IgG (H+L) coupled to HRP for 1 h at room temperature. After three washes with PBS-T pH7,20.1% Tween-20 for 3x5 min at room temperature, bands from each individual serum incubation are taped onto a plastic sheet.

The slide is then incubated with ECL substrate (Biorad Clarity) to visualize bound human IgG on a BioRad WesternBlot detection system. The signals corresponding to the amount of IgGs that specifically bind to the Rep band of the protein membrane are quantified by densitometry.

An example of a 1D size resolved Rep protein membrane band result is shown in Figure 1 and quantification of seropositive serum/plasma samples is shown in Table 2: Almost 50% of the serum samples gave signals very strong for both dilutions (1:500 and 1:2000) and only 3 of 21 were totally negative. Of the plasma controls, 7 samples gave intermediate signals at the 1:500 dilution (some additional with very low signals) while at the 1:2000 dilution only 3 samples gave a very weak signal, while all the others were seronegative. All quantifications support the trend that MS-serum intensities show at least a 30-fold excess of signal when compared to control plasma intensities.

Table 2: Quantification of seropositive serum/plasma samples.

Sera/plasma with Rep-specific human IgG detection were counted according to signal intensity for the serum/plasma dilution of 1:500 and 1:2000 for the MS serum group and the healthy control plasma group. (each 21 in total). Almost 50% of the serum samples gave very strong signals for both dilutions (1:500 and 1:2000) and only 3 of 21 were totally negative. Of the plasma controls, 7 samples gave intermediate signals at the 1:500 dilution (some additional ones with very low signals) while at the 1:2000 dilution only 3 samples gave a very weak signal, while all the others were seronegative. All quantifications support the trend that MS-serum intensities show at least a 30-fold excess of signal when compared to control plasma intensities.

Example 4:

Serum detection by ELISA

An appropriate amount of purified Rep protein (typically between 50 and 200 ng per well) is added to a 1:1 mixture of 8 M urea pH 8.0 and PBS pH 7.2 predisposed in a 96-well Maxisorp ELISA plate (Fisher scientific). The protein is allowed to bind to the plate matrix for 14 h at 4°C on a linear shaker. The plate is then washed with PBS-T pH 7.2 0.1% Tween-20 for 3x5 min at room temperature, followed by blocking for at least 14 h at 4°C with PBS pH 7.2 1%(w/ v) BSA finally containing 0.02% sodium azide. Serum is then added at a 1:500 dilution and incubated for 6 h at room temperature. The plate is then washed with PBS-T pH 7.20.1% Tween-20 for 3x5 min at room temperature. Detection of human IgG specifically bound to Rep in sera is performed by incubation with an HRP-coupled goat anti-human IgG secondary antibody (H+L) for 1 h at room temperature. After three washes with PBS-T pH 7.20.1% Tween-20 for 3x5 min at room temperature, 100 µl of TMB substrate solution (3,3',5,5'-tetramethylbenzidine, sensitive to HRP) per well and incubate for 5 min at room temperature. The reaction is stopped by adding 100 pl of 8M acetic acid/1M sulfuric acid. Signal intensity is quantitated on an ELISA-compatible Perkin Elmer device by measuring absorbance at 450 nm.

The result of an example of quantification of Rep-specific sero-responses based on ELISA screening is shown in Figure 2: Quantitation reveals a good correlation between signal intensity and the amount of Rep protein (antigen). On average, the MS group's signal intensity levels exceeded the control group's intensities by at least a factor of 1.9. In general, ELISA-based serum screening revealed detectable antibodies in serum to at least a 1:1000 dilution of serum (data not shown), so it is likely that the titer for this experiment is greater than 1:1000 in the region of 1:2000-1:4000 or even higher after platform optimization.

Example 5:

Immunofluorescence analysis

HEK293TT cells (125,000/well) were cultured for 24 h in 8-well immunofluorescence chambers in DMEM 10% FCS (supplemented with 1* Glutamax (Gibco) and 1* non-essential amino acids (Gibco)) followed by transfection. Polyethyleneimine (PEI) DNA sample of the control plasmid ZsGreenlCI (Clontech) expressing the autofluorescence marker protein ZsGreen or the same plasmid with the complete MSBI1 Rep ORF at the 3-prime end encoding a ZsGreen-Rep fusion protein. 48 hours after DNA transfection, cells were washed with PBS, fixed for 20 min at Rt with 4% PFA in PBS, and permeabilized with 0.5% Triton X-100 in PBS for 10 min at RT. in a stirring device. Cells were washed with PBS and blocked with PBS 1% BSA for 45 minutes at room temperature. Incubation with the mouse anti-Rep monoclonal antibodies (c.f. Table 1) was performed for 1 h at 37 °C in PBS 1% BSA with 1:500 antibody dilutions (controls without primary antibodies were included). Cells were washed three times with PBS followed by incubation with PBS 1% BSA for 15 min at RT. Next, incubation with the secondary antibody (goat anti-mouse AlexaFluor546, 1:500; Hoechst 33342 DNA marker, 1:5,000) was performed for 45 min at RT in PBS 1% BSA. Cells were washed three times with PBS 1% BSA and three times with PBS before mounting with coverslips (Dako mounting medium). Immunofluorescence images were taken on a Zeiss Cell Observer (20x objective, fixed exposure time for sample and control cubes).

Immunofluorescence images (Fig. 4) show that treatment with group A and C antibodies leads to specific detection of target protein located in cytoplasmic nuclear membrane (+nucleus) without detection of aggregates. In contrast, group B and D antibodies show specific colocalization with the speckle protein and no or weak levels of cytoplasmic signals. Control incubation of antibodies on ZsGreen fusion protein alone did not result in detection of significant signals (same exposure times used for visualization of antibody signals).

Example 6:

Immunofluorescence analysis

HEK293TT cells (125,000/well) were cultured for 24 h in 8-well immunofluorescence chambers in DMEM 10% FCS (supplemented with 1* Glutamax (Gibco) and 1* non-essential amino acids (Gibco)) followed by DNA transfection with polyethyleneimine (PEI) of the control plasmid ZsGreenlCI (Clontech) expressing the autofluorescence marker protein ZsGreen or the same plasmid with the complete CMI1 Rep ORF at the 3-prime end encoding a ZsGreen-Rep fusion protein . 48 hours after DNA transfection, cells were washed with pBs, fixed for 20 min at R ^t with 4% PFA in PBS, and permeabilized with 0.5% Triton X-100 in PBS for 10 min. at RT in a stirring device. Cells were washed with PBS and blocked with PBS 1% BSA for 45 minutes at room temperature. Incubation with the anti-Rep mouse monoclonal antibodies (Table 1) was performed for 1 h at 37 °C in PBS 1% BSA with 1:500 antibody dilutions (controls without primary antibodies were included). Cells were washed three times with PBS followed by incubation with PBS 1% BSA for 15 min at RT. Next, incubation with the secondary antibody (goat anti-mouse AlexaFluor546, 1:500; Hoechst 33342 DNA marker, 1:5,000) was performed for 45 min at RT in PBS 1% BSA. Cells were washed three times with PBS 1% BSA and three times with PBS before mounting with coverslips (Dako mounting medium). Immunofluorescence images were taken on a Zeiss Cell Observer (20x objective, fixed exposure time for sample and control doublets).

Immunofluorescence images (Fig. 5) show that while the group A antibody specifically detects target protein localized in the cytoplasm nuclear membrane (+nucleus) without detection of aggregates, the group B antibody detects speckled target protein with minimal protein background. target located in cytoplasm. Group C and D MSBI1-specific antibodies do not lead to specific detection of signals. Both D1 and D2 antibodies belong to group "D" but have slightly different epitope recognition.

Example 7:

Immunofluorescence analysis

HEK293TT cells (125,000/well) were cultured for 24 h in 8-well immunofluorescence chambers in 10% DMEM FCS (supplemented with 1*Glutamax (Gibco) and 1*non-essential amino acids (Gibco)) followed by DNA transfection with polyethyleneimine (PEI) from the control plasmid pcDNA3.1(-) (Invitrogen) or from the same plasmid expressing a codon-optimized variant of the complete ORF of MSBI1 Rep (SEQ ID. No.: 13). 48 hours after DNA transfection, cells were washed with PBS, fixed for 20 min at RT with PBS 4% PFA at RT, and permeabilized with PBS 0.5% Triton X-100 for 10 min at TA in a stirring device. Cells were washed with PBS and blocking was performed with PBS 1% BSA for 45 min at room temperature. Incubation with monoclonal anti-Rep antibodies mouse (Table 1) was performed for 1 h at 37 °C in PBS 1% BSA with 1:500 antibody dilutions (controls lacking primary antibodies were included). Cells were washed three times with PBS followed by incubation with 1% pBs BSA for 15 min at room temperature. Then, incubation with the secondary antibody (goat anti-mouse AlexaFluor488, 1:500; Hoechst 33342 DNA marker, 1:5,000) was performed for 45 min at RT in PBS 1% BSA. Cells were washed three times with PBS 1% BSA and three times with PBS before mounting with coverslips (Dako mounting medium). Immunofluorescence images were taken on a Zeiss Cell Observer (20x objective, fixed exposure time for sample and control doublets).

Immunofluorescence images (Fig. 6) show that antibody treatment with group A and C antibodies leads to specific detection of target protein located in cytoplasmic nuclear membrane (+nucleus) without detection of aggregates. In contrast, group B and D antibodies show specific colocalization with speckled protein and no or weak levels of cytoplasmic signals. Control incubation of antibodies in ZsGreen fusion protein alone did not result in significant signal detection (same exposure times used for visualization of antibody signals).

Example 8:

Western blot analysis

HEK293TT (1.5 Mio/6 wells) were cultured for 24 h in 6-well cell culture plates in DMEM 10% FCS (supplemented with 1* Glutamax (Gibco) and 1* non-essential amino acids (Gibco)) followed by transfection. Polyethyleneimine (PEI) DNA screening of ZsGreenlCI plasmids encoding overexpression of the full-length MSBI1, CMI1 or MSBI2 Rep protein as a fusion protein with an N-terminal ZsGreen tag. 48 hours after DNA transfection, cells were washed with PBS, trypsinized, washed again with PBS, and lysed in SDS-PAGE Lammli buffer (boil for 5 min at 98 °C). Samples were loaded onto precast 12.5% SDS-PAGE gels with a large pocket. After the transfer blot, two strips of the membrane were cut out to be individually incubated with the different mouse monoclonal antibodies from Table 1 (1:1000 dilution in PBS 5% skim milk) after blocking with PBS 5% skim milk. The strips were washed three times with PBS 0.1% Tween20 and incubated with the HRP-coupled anti-mouse secondary antibody allowing Western blot analysis of antibodies detecting the different ZsGreen-Rep fusion proteins on a Biorad ChemiDoc device. The detection of the positive control was made from the incubation with a ZsGreen antibody (OriGene, 1:2000)

Fig. 7 shows that while the antibodies from groups A and B specifically detect all three Rep fusion proteins, including the Rep fusion proteins CMI1 and MSBI2 in addition to the Rep fusion protein MSBI1, the MSBI1-specific antibodies from groups C and D exclusively detect the Rep MSBI1 fusion protein. The D2 antibody is WB-negative most likely due to the detection of a 3D structure epitope, which does not prevail under denaturing conditions on SDS-PAGE and was not analyzed here.

SEQUENCE SUMMARY







 REFERENCES:

Funk, M., et al. (2014). "Isolation of protein-associated circular DNA from healthy cattle serum". Genome Announcement 2(4)

Giraldo, R., et al. (2011). "RepA-WH1 prionoid: a synthetic amyloid proteinopathy in a minimalist host." Prion 5(2):60-64

Gunst, K., et al. (2014). "Isolation of bacterial plasmid-related replication-associated cirular DNA from a serum sample of a multiple sclerosis patient." Genome Announcement 2(4).

Lamberto, I., et al. (2014). "Mycovirus-like DNA virus sequences from cattle serum and human brain and serum samples from multiple sclerosis patients." Genome Announcement 2(4).

Manuelidis L., 2011. "Nuclease resistant circular DNAs co-purify with infectivity in scrapie and CJD". J. Neurovirol. 17:131-145.

Torreira, E., et al. (2015). "Amyloidogenesis of bacterial prionoid RepA-WH1 recaptiulates dimer to monomer transitions of RepA in DNA replication initiation." Structure 23(1):183-189

Whitley, C., et al. (2014). "Novel replication-competent cirulara DNA molecules from healthy cattle serum and milk and multiple sclerosis-affected human brain tissue." Genome Announcement 2(4).

Claims (15)

1. A method for diagnosing multiple sclerosis (MS), in which (A) at least 2-fold greater amount of a DNA replication-associated protein (Rep) or fragments thereof having at least 8 contiguous amino acids in a sample from a subject compared to an amount in a control sample ; O well (B) at least 2-fold greater amount of anti-Rep antibodies in a sample from a subject compared to an amount in a control sample correlates with a diagnosis of MS, in which — the Rep protein comprises (i) an amino acid sequence as described in NO. ID. SEQ. :1; (ii) a fragment of NO. ID. SEQ. :1 which is capable of binding to an anti-Rep antibody specific for a protein having the amino acid sequence of NO. ID. SEQ. 1; either (iii) an amino acid sequence that is 90% or more homologous to the amino acid sequence of (i) or (ii) and is capable of binding to an anti-Rep antibody specific for a protein having an amino acid sequence not. ID. SEQ. :1, and — the sample is (a) a liquid sample selected from serum, plasma or cerebrospinal fluid or (b) a solid sample selected from tissue culture or biopsy samples. The method of claim 1, wherein the NO. ID. SEQ. :1 comprises an epitope within the amino acid sequence consisting of amino acids 1 to 229 of NO. ID. SEQ. :1. The method of claim 1 or 2, wherein an increased amount of Rep protein or fragments thereof or anti-Rep antibodies of at least 5-fold compared to a control sample indicates MS. The method of claim 1, wherein the Rep protein is encoded by a polynucleotide acid as represented by NO. ID. SEQ. :13. 5. An anti-Rep antibody selected from the group consisting of DSM ACC3327 antibody (AB01 523-1-1), DSM ACC3328 antibody (AB02 304-4-1), DSM ACCC3329 antibody (MSBI1 381-6-2 ), the DSM ACC 3330 antibody (MSBI761-5-1) and the DSM ACC 3331 antibody (MSBI1 961-2-2). 6. Use of an anti-Rep antibody of claim 5 in a method of any of claims 1 to 4. 7. A method for diagnosing MS in a subject comprising the steps of (a) incubating a sample selected from a serum or plasma sample obtained from a subject with Rep protein as defined in claim 1; (b) detecting the amount of antibodies in the subject's sample that form an immune complex with the Rep protein; and (c) correlating at least a 2-fold higher amount of antibodies bound to the Rep protein in the subject's sample, compared to an amount in a control sample, with a diagnosis of MS. The method of claim 7, wherein in step (a) the Rep protein is immobilized and the immobilized Rep protein is then incubated with the subject's sample. The method of claim 7, wherein in step (a) the Rep protein is expressed in cells followed by incubation of the cells with the subject's sample. The method of claim 8 or 9, wherein in step (b) the amount of antibodies forming an immune complex with the Rep protein is quantitated by a detector binding agent coupled to a signal generating compound. The method of claim 7, wherein in step (a) the subject sample antibodies are immobilized followed by incubation with a defined amount of Rep protein. 12. A method of diagnosing MS in a patient comprising the steps of (a) detecting the amount of Rep protein in a sample selected from a subject's serum, plasma or tissue sample by anti-Rep antibodies that bind to an epitope comprised of NO. ID. SEQ. :2 or NO. ID. SEQ. :3, and (b) correlating at least a 2-fold greater amount of Rep protein detected in the sample from a subject in step (a) compared to an amount in a control sample with a diagnosis of MS. The method of claim 12, wherein the antibody specific for the Rep protein binds to an epitope that falls within an amino acid sequence selected from the group consisting of amino acids 1 to 136, 137 to 229 and from 230 to 324 of NO. ID. SEQ. :1. 14. A kit for use in the diagnosis of MS comprising (a) a Rep protein, wherein the Rep protein comprises: (i) an amino acid sequence as represented by NO. ID. SEQ. :1; (ii) a fragment of NO. ID. SEQ. :1 that is capable of binding an anti-Rep antibody with specificity for a protein having the amino acid sequence of NO. ID. SEQ.: 1; or (iii) an amino acid sequence that is 90% or more homologous to the amino acid sequence of (i) or (ii) and is capable of binding to an anti-Rep antibody with specificity for a protein having the sequence of NO amino acids. ID. SEQ. :1, (b) an anti-human antibody coupled to a detectable label selected from the group consisting of DSM ACC3327 antibody (AB01 523-1-1), DSM ACC3328 antibody (AB02 304-4-1), DSM ACCC3329 antibody (MSBI1 381- 6-2), the DSM ACC 3330 antibody (MSBI761-5-1) and the DSM ACC 3331 antibody (MSBI1 961-2-2), and (c) a solid matrix suitable for immobilizing a Rep protein according to (a) or anti-Rep antibodies with specificity for a protein having the amino acid sequence of NO. ID. SEQ.: 1 suspected in a serum or plasma sample. The kit according to claim 14 for use in an assay selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), and strip test.